Multipartite high-affinity nucleic acid probes

ABSTRACT

The invention provides a collection of probes useful for hybridizing to a target nucleic acid. The probes associate with each other, binding with high affinity to the target nucleic acid, to form three-way junctions and other complexes. At least one of the probes in each collection includes a nucleic acid analog. Methods using the probes in hybridization and as primers are also provided.

I. FIELD OF THE INVENTION

The present invention generally relates to the fields of nucleic acidanalogs and hybridization. More specifically, the invention relates tomethods and compositions for hybridization of a collection of probes toa target nucleic acid.

II. BACKGROUND

Nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid(RNA), are bearers of information. This information, encoded in theordered nucleotides that constitute the nucleic acids, enables a livingsystem to construct a protein, a cell, or an organism. One specificsequence of nucleotides may be found in a virus, whereas a differentsequence may be found in a bacterium, and yet a different sequence in ahuman being. The detection and analysis of nucleic acids has become oneof the most fundamental aspects of diagnostic medicine and medicalresearch. Because nucleic acid sequences can also differ amongindividuals, nucleic acid analysis has also become important to forensicmedicine.

Most current methods of nucleic acid analysis require the hybridizationof one or more oligonucleotides to a target nucleic acid of interest.The hybridization step is often followed by an enzymatic reactioninvolving the addition of nucleotides to the hybridized oligonucleotide,as in primer extension reactions or in polymerase chain reactions (PCR).In other cases, the hybridization step is followed by washing anddetection steps, as in Southern or Northern blot analysis.

The hybridization of an oligonucleotide to a target nucleic acidgenerally requires that the sequence of the oligonucleotide beapproximately complementary to the sequence of the target nucleic acid.Thus, after the sequence of the target nucleic acid of interest isdetermined, an appropriate, complementary oligonucleotide for use inhybridization to the target nucleic acid must be designed.

In most cases, the complementary oligonucleotide must becustom-synthesized. Most oligonucleotides used for this purpose are atleast twelve nucleotides in length to permit efficient hybridization.Because any of four nucleotides could be present at each position in theoligonucleotide, there are 4¹² or 16,777,216 possible oligonucleotidesthat are twelve nucleotides in length. The skilled artisan is thereforeunlikely to possess, in advance, a newly-desired oligonucleotide.Unfortunately, custom synthesis of oligonucleotides is both expensiveand time-consuming: the process may require from 3-6 business days,including ordering, synthesis, and shipping. Inevitably, analysis of thenucleic acids is further delayed.

Thus, because of the importance of nucleic acid analysis to modemmedical science, there is a great need for faster, cheaper, and morereliable means to carry out this analysis, such as methods that do notdepend on custom-synthesized oligonucleotides. Similarly, there is aneed for nucleic acid binding moieties that can be generated rapidly andinexpensively without the impediments of traditional custom synthesis.

III. SUMMARY OF THE INVENTION

It has been discovered that a nucleic acid binding moiety can begenerated by combining two or more smaller probes that interact togenerate a single binding moiety, also referred to herein as amultipartite binding moiety. For example, if one of the smaller probesincludes a portion complementary to a first nucleic acid sequence (“X”),and another of the smaller probes includes a portion complementary to asecond nucleic acid sequence (“Y”), then a new, single binding moietywould include a composite nucleic acid recognition sequencecomplementary to the combination of the first and second regions (X+Y).Accordingly, the invention allows one skilled in the art to create aprobe complementary to a target nucleic acid sequence by combiningsmaller probes, each of which is complementary to a particular subset ofthe target nucleic acid sequence and is capable of interacting with theother smaller probe(s). This interaction can be achieved, for example,through the formation of a three-way junction.

It has been discovered that a three-way junction can be stabilized bythe introduction of a flexible linker between the portion of the smallerprobe that interacts with the target nucleic acid and the portion thatinteracts with the other smaller probe(s). It has also been discoveredthat the use of peptide nucleic acids or other nucleic acid analogs thatinteract more strongly with a strand of DNA than would the complementarystrand of DNA can improve the affinity of the smaller probes for eachother and for the target nucleic acid. Thus, a stable interaction ispossible even where each of the smaller probes is complementary only toa small region of the target nucleic acid (e.g. three to eightnucleotides).

One aspect of the invention is a collection of at least two probes foruse in hybridizing to a target nucleic acid. In this aspect, a firstprobe includes a first portion that may be complementary to a firstregion of a target nucleic acid and capable of hybridizing thereto,joined by a flexible linker to a second portion capable of hybridizingwith the second probe. Similarly, the second probe includes a firstportion that may be complementary to a second region of the targetnucleic acid and capable of hybridizing thereto, and a second portioncapable of hybridizing with the first probe. Both the first and secondregions of the target nucleic acid typically are from three to eightnucleotides in length, and preferably substantially adjacent, i.e.separated by zero or one nucleotides. Either the first probe or thesecond probe, or both, is or includes a high-affinity nucleic acidanalog. A preferred high-affinity nucleic acid analog is PNA (peptidenucleic acid), where the sugar/phosphate backbone of DNA or RNA has beenreplaced with a polyamide backbone, e.g. 2-aminoethylglycine.

Because this invention provides a means to generate a larger probe bycombining two or more smaller probes, in other embodiments of theinvention, the collection of probes includes an array of a plurality offirst probes (a library of first probes) and an array of a plurality ofsecond probes (a library of second probes) which may be used to generatea larger probe. In these embodiments, the portion of each of the firstprobes that may be complementary to the first region of the targetnucleic acid has a different sequence, and the portion of each of thesecond probes that may be complementary to the second region of thetarget nucleic acid has a different sequence.

The array of first probes often includes at least 50% of the possiblecombinations of first probes. Thus, if the first region of the targetnucleic acid is x nucleotides in length and if the portion of each ofthe first probes that may be complementary to that region isnon-degenerate, the array of first probes includes at least 0.5×4^(x)first probes. Likewise, the array of second probes often includes atleast 50% of the possible combinations of second probes.

In other embodiments, the collection of probes is provided in a kit, incombination with a buffer. In one preferred embodiment, the kit alsoincludes an enzyme. In another preferred embodiment, the kit alsoincludes a detection moiety.

Another aspect of the invention is a method of using the collection ofprobes of the invention. In one embodiment, the invention is a method ofdetecting the presence of a target nucleic acid sequence. Generally, thetarget nucleic acid is exposed to the first and second probes to form acomplex if the target nucleic acid sequence is present. The presence orabsence of the complex may be determined by fluorescent assays,colorimetric assays, enzymatic assays, or by any other means capable ofdetecting the presence or the absence of the complex. In anotherembodiment, the exposure and detection steps are iterated usingdifferent combinations of first and second probes derived from an arrayof a plurality of first probes and an array of a plurality of secondprobes.

In another embodiment, the invention is a method of priming anenzyme-catalyzed reaction such as polymerase chain reaction (PCR),primer extension, ligation, or other amplification methods. In thisembodiment, the target nucleic acid is exposed to the first and secondprobes under conditions that permit the formation of a complex if thetarget sequence in the target nucleic acid is present. In thisembodiment, an enzyme typically is provided to catalyze a reactionprimed by the complex.

It should be understood that the order of the steps of the methods ofthe invention is immaterial so long as the invention remains operable,i.e. permits the formation of a multipartite binding moiety, itsassociation with a target nucleic acid, and any subsequent operations orsteps. Moreover, two or more steps may be conducted simultaneously.

The foregoing, and other features and advantages of the invention, aswell as the invention itself, will be more fully understood from thefollowing description, drawings, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show schematic depictions of multipartite binding moietieshybridizing to target nucleic acids. FIG. 1A shows one embodiment of athree-way junction. FIG. 1B shows one embodiment of a four-way junction.FIG. 1C shows one embodiment of a pair of three-way junctions. Solidlines represent target and probe. Dashed lines represent associations,e.g. Watson-Crick base pairing. Gray curved lines are flexible linkers.

FIGS. 2A and 2B show structures of fluorescent dye moieties that may beuseful in labelling probes and nucleotides. The attachment site of thedye, X, is linked to a probe or nucleotide.

FIG. 3 shows structures of quenching moieties that may be useful in FRETlabelling of probes. Substituents Z may be alkyl, aryl, or functionalgroup. The attachment site of the quencher, X, is linked to a probe ornucleotide.

FIGS. 4A-4D show schematic depictions of probes bearing fluorescent andquenching moieties that may be useful in the practice of one aspect ofthe invention. In FIGS. 4A and 4B, the fluorescent and quenchingmoieties are covalently attached to the same probe. In FIGS. 4C and 4D,the fluorescent and quenching moieties are covalently attached todifferent probes. In FIGS. 4A and 4C, the probe(s) is (are) notassociated with a target nucleic acid, and the fluorescent and quenchingmoieties are in proximity with each other and quenching occurs by FRET.In FIGS. 4B and 4D, the probe(s) is (are) associated with a targetnucleic acid in a three-way junction, and the fluorescent and quenchingmoieties are separated, permitting fluorescence and detection of thecomplex.

FIG. 5 schematically depicts an embodiment of a method of generating abinding moiety by selecting probes from libraries of the presentinvention.

FIG. 6 schematically illustrates the conceptual structure of thelibraries of the present invention by an exemplary depiction ofdinucleotide regions of probes in libraries and formation of an adjacentthree-way junction with a target sequence complementary to the twoselected dinucleotide-containing probes.

FIG. 7 shows a three-way junction formed from fluorescein-labelled (Flu)ME01 PNA, rhodamine-labelled (Rho) ME02 PNA, and 1057 wild-type DNA.

FIG. 8 shows the digitized image of gel electrophoresis (10%polyacrylamide) conducted under native, non-denaturing conditions, i.e.room temperature and no urea. Fluorescence detection.

FIG. 9 shows the digitized image of gel electrophoresis (10%polyacrylamide) of the same samples as FIG. 8, conducted underdenaturing conditions, i.e. 35° C. and 7M urea. Fluorescence detection.

FIG. 10 shows the PNA probes and DNA oligonucleotides used in themultipartite and control complex analyses in each lane of FIGS. 8 and 9.

FIG. 11 shows the sequences of the PNA probes and DNA oligonucleotidesused in the multipartite and control complex analyses in each lane ofFIGS. 8 and 9. Flu -carboxyfluorescein. Rho-tetramethylrhodamine(TAMRA). O-O linker, linker (2-[2-(2- aminoethoxy]acetic acid.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS V.1. Definitions

As used herein, “nucleic acid” encompasses the terms oligonucleotide andpolynucleotide and denotes single stranded and double-stranded polymersof nucleotide monomers, including 2′-deoxyribonucleotides (DNA) andribonucleotides (RNA). The nucleic acid may be composed entirely ofdeoxyribonucleotides, entirely of ribonucleotides, or chimeric mixturesthereof. The monomers are typically linked by internucleotidephosphodiester bond linkages, and associated counterions, e.g., H⁺, NH₄⁺, trialkylammonium, Mg²⁺, and Na⁺. Nucleic acids typically range insize from a few monomeric units, e.g., 5′40, when they are commonlyreferred to as oligonucleotides, to several thousands of monomericunits. Whenever an oligonucleotide sequence is represented, it will beunderstood that the nucleotides are in 5′to 3′order from left to rightand that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G”denotes deoxyguanosine, and “T” denotes thymidine, unless otherwisenoted.

As used herein, “nucleic acid analog” is understood to mean a structuralanalog of DNA or RNA, designed to hybridize to complementary nucleicacid sequences (Hunziker, J. and Leumann, C. (1995) “Nucleic AcidAnalogues: Synthesis and Properties” in Modern Synthetic Methods, Vol.7, pp. 331-417). Through modification of the internucleotide linkage(s),the sugar, and/or the nucleobase, nucleic acid analogs may attain any orall of the following desired properties: 1) optimized hybridizationspecificity or affinity, 2) nuclease resistance, 3) chemical stability,4) solubility, 5) membrane-permeability, and 6) ease or low costs ofsynthesis and purification. Examples of nucleic acid analogs include,but are not limited to, peptide nucleic acids (PNA), locked nucleicacids “LNA” (Imanishi, etal WO 98/39352; Imanishi, etal WO 98/22489;Wengel, etal WO 00/14226), 2′-O-methyl nucleic acids (Ohtsuka, etal,U.S. Pat. No. 5,013,830), 2′-fluoro nucleic acids, phosphorothioates,and metal phosphonates.

The term “target nucleic acid” are capable of hybridizing with theprobes of the invention and include genomic DNA, DNA digests, plasmids,vectors, viral DNA, PCR products, RNA, and synthetic nucleic acids.Target nucleic acid may also be a metaphase or interphase chromosome.Target nucleic acids may be single-stranded or double-stranded and canrange from as few as about 20-30 nt to as many as millions ofnucleotides (nt) or base-pairs (bp), depending on the particularapplication. “Target sequence” means a polynucleotide sequence that isthe subject of hybridization with a complementary polynucleotide, e.g. aprimer or probe. The sequence can be composed of DNA, RNA, an analogthereof, including combinations thereof. The terms “target region”,“target sequence”, and “target nucleic acid sequence” mean a region of anucleic acid which is to be detected.

As used herein, “high-affinity nucleic acid analog” is understood tomean a nucleic acid analog with a higher binding affinity for a strandof DNA than the corresponding complementary strand of DNA. Examplesinclude PNA, LNA, 2′-O-methyl nucleic acids, and 2′-fluoro nucleicacids. Examples of nucleic acid analogs also include chimera moleculescomprising one or more nucleic acid analog units and one or more DNA(2′-deoxynucleotide) units. For example, a nucleic acid analog may be aPNA-DNA chimera comprised of a PNA moiety and a DNA moiety.

As used herein, “PNA” or “peptide nucleic acid” is understood to mean ahigh-affinity nucleic acid analog in which the sugar/phosphate backboneof DNA or RNA has been replaced with a polyamide-based backbone.

As used herein, “flexible linker” is understood to mean a region of aprobe that is conformationally more flexible than a strand of DNA.Preferably, a flexible linker minimizes strain in a three-way orhigher-order junction. Appropriate flexible linkers include, but are notlimited to, ethyleneoxy units and alkyldiyl. “Detection moiety” means alabel enabling the detection of a complex. Preferably, the detectionmoiety permits the direct detection of the complex. Detection moietiesinclude, but are not limited to, fluorescent moieties, enzymaticmoieties, or moieties comprising a defined antigen (e.g., ahemagluttinin tag, a myc tag, or the like). “Destabilizing moiety” meansa label, functional group or other modification to one or more of theprobes in a multipartite complex that effectively decreases the meltingtemperature of the complex formed by the coming together of two or moreprobes, typically by destabilizing base pairing and hydrogen bonding asusually occurs when complementary strands of nucleic acids becomehybridized.

“Probe” means a nucleic acid or nucleic acid analog which is useful forhybridization to a target nucleic acid. For example, a probe may be afirst nucleic acid or nucleic acid analog connected to a second nucleicacid or nucleic acid analog by a flexible linker. A probe may optionallyinclude a detectable moiety.

As used herein, “substantially adjacent” describes two regions of anucleic acid that are separated by zero or one intervening nucleotides.

As used herein, a “universal” nucleobase is understood to mean anucleobase that does not discriminate among cytosine, guanine, adenine,thymine, and uracil. Universal nucleobases include, for example,xanthine and 5-nitropyrole (Nature (1994) 369:492-493).

As used herein, “degeneracy” is understood to mean the number ofnaturally-occurring nucleotide sequences to which a given probe iscomplementary. Thus, a non-degenerate probe has a degeneracy of one,whereas a probe with one universal nucleobase has a degeneracy of four,and a probe with two universal nucleobases has a degeneracy of sixteen.

V.2. Structure of the Probes—a First Portion for Binding to a TargetNucleic Acid

A multipartite nucleic acid binding moiety is formed by the combinationof at least two smaller probes, each of which can interact both directlywith a target nucleic acid and directly or indirectly with each other.The binding moiety can interact with a target nucleic acid, and thespecificity of the binding moiety for the target nucleic acid isdetermined by the combined specificities of certain sequences of thesmaller probes that form the binding moiety. Thus, the skilled artisancan rapidly generate a binding moiety complementary to a particulartarget nucleic acid by judiciously selecting smaller probes andcombining them to form a single binding moiety. This invention thereforefacilitates and accelerates nucleic acid analysis and is effective tosave time and money in medical research, diagnostic medicine, forensicmedicine, and in all other branches of biology and medicine requiringthe study of nucleic acids.

The invention relates to a binding moiety composed of at least twoprobes. Each probe comprises a first portion that may be complementaryto a region of a target nucleic acid and capable of hybridizing thereto.For example, the binding moiety 10 in FIG. 1A is composed of a firstprobe 12 and a second probe 14. The first probe 12 contains a firstportion 16 complementary to a first region 18 of the target nucleic acid20. Similarly, the second probe 14 contains a first portion 22complementary to a second region 24 of the target nucleic acid 20. Thefirst portions 16 and 22 include a nucleic acid, nucleic acid analog, ora chimera thereof. The presence of a nucleic acid in at least one of thefirst portions 16 and 22 is preferred in some embodiments, particularlywhere binding of the probes to the target nucleic acid 20 is followed byan enzymatic reaction as discussed below. The first probe and/or thesecond probe comprise a high-affinity nucleic acid analog. The bindingmoiety 10 and target 20 may form a three-way junction, as shown in FIG.1A. Three-way junctions and higher-order nucleic acid complexes havebeen studied. See for example: Duckett etal (1990) EMBO Journal,9:1659-64; Leontis etal (1993) J. Biomol. Structure & Dynamics,11:215-23; Rosen etal (1993) Biochemistry, 32:6563-87; Shlyakhtenko etal(1994) J. Biomol. Structure & Dynamics, 12:131-43; Husler etal (1995)Arch. of Biochemistry and Biophysics 322:149-66; Welch etal (1995) J.Mol. Biol. 251:507-19; Yang etal (1996) Biochemistry 35:7959-67.

In most embodiments, the presence of a high-affinity nucleic acid analogin at least one of the first portions 16 and 22 is preferred. In a morepreferred embodiment, each of the first portions includes ahigh-affinity nucleic acid analog. A particularly preferredhigh-affinity nucleic acid analog is PNA. For example, PNA in which thesugar/phosphate backbone of DNA or RNA has been replaced with2-aminoethylglycine demonstrates exceptional hybridization specificityand affinity when nucleobases are attached to the linkage through anamide bond (WO 92/20702; P. Nielsen etal., “Sequence-selectiverecognition of DNA by strand displacement with a thymidine-substitutedpolyamide,” Science (1991) 254:1497-1500). Synthesis of PNA can beconducted by any method known in the art (U.S. Pat. Nos. 5,539,082,5,714,331, 5,786,461; also WO 93/12129).

2-Aminoethylglycine PNA oligomers typically have greater affinity, i.e.hybridization strength and duplex stability, for their complementaryPNA, DNA, and RNA than the corresponding DNA sequences (U.S. Pat. No.5,985,563; WO 98/24933; WO 99/22018; WO 99/21881; WO 99/49293). Themelting temperatures (T_(m)) of PNA/DNA and PNA/RNA hybrids are higherthan those of the corresponding DNA/DNA or DNA/RNA duplexes (generally1° C. per bp) due to a lack of electrostatic repulsion in thePNA-containing complexes. Also, unlike DNA/DNA duplexes, the T_(m) ofPNA/DNA duplexes are largely independent of salt concentration.

2-Aminoethylglycine PNA oligomers also demonstrate a high degree ofbase-discrimination (specificity) in pairing with their complementarystrand. Specificity of hybridization can be measured by comparing T_(m)values of duplexes having perfect Watson/Crick complementarity withthose having one or more mismatches. The degree of destabilization ofmismatches, measured by the decrease in T_(m) (ΔT_(m)), is a measure ofspecificity. In addition to mismatches, specificity and affinity areaffected by structural modifications, hybridization conditions, andother experimental parameters (Egholm, etal (1993) Nature 365:566-68).

Thus, by including PNA or other high-affinity nucleic acid analogs infirst portion(s) 16 and/or 22, specificity of binding is maintained orincreased, and the affinity for the target is increased. Thus, use ofPNA or other high-affinity nucleic acid analogs permits the use ofsmaller first portions of probes without sacrificing affinity. Forexample, whereas a DNA oligonucleotide might require binding to 15 ormore nucleotides of a target nucleic acid for stable interaction, a PNAmay require binding to less than 15 nucleotides under the sameconditions. Preferably, neither the first probe 12 nor the second probe14 contains a destabilizing moiety (Weston etal, WO 99/37806).

Accordingly, in a preferred embodiment, a first portion 16 of a firstprobe 12 is complementary to a first region 18 of from about 3 to 8nucleotides in length. As shown as an example in FIG. 1A, first portion16 binds to a six nucleotide first region 18 of target 20.

Preferably, the first portion 22 of a second probe 14 is complementaryto a second region 24 of from 3 to 8 nucleotides in length. Morepreferably, the second region 24 is from five to seven nucleotides inlength. In another preferred embodiment, the combined lengths of thefirst and second regions are from 6 to 12 nucleotides.

The neutral backbone of PNA also increases the rate of hybridizationsignificantly in assays where either the target, template, or the PNAprobe is immobilized on a solid substrate. Without any electrostaticrepulsion, the rate of hybridization is often much higher for PNA probesthan for DNA or RNA probes in applications such as Southern blotting,Northern blots, or in situ hybridization experiments (D. Corey, (1995)“48,000-fold acceleration of hybridization by chemically modifiedoligonucleotides,” J. Amer. Chem. Soc. 117:9373-74).

With certain DNA sequences, a second PNA can further bind to form anunusually stable triple helix structure (PNA)₂/DNA. PNA have beeninvestigated as potential antisense agents, based on theirsequence-specific inhibition of transcription and translation (Lee etal,(1998) Biochemistry 37:900-10; Nielsen, P. (1996) Antisense Therapeutics4:76-84). However, PNA oligomers themselves are not substrates for apolymerase as primers or templates, and do not conduct primer extensionwith nucleotides (U.S. Pat. No. 5,629,178).

Accordingly, if primer extension or similar enzyme-catalyzed reactionsare to follow binding of the probes to a target nucleic acid, at leastone of the probes preferably includes a PNA-DNA chimera with a 3′hydroxyl. PNA-DNA chimeras are oligomer molecules with discrete PNA andnucleotide moieties. They can be synthesized by covalently linking PNAmonomers and nucleotides in virtually any combination or sequence.Efficient and automated methods have been developed for synthesizingPNA-DNA chimeras (Vinayak etal., (1997) Nucleosides & Nucleotides16:1653-56; Uhlmann etal (1996) Angew. Chem., Intl. Ed. Eng. 35:2632-35;Van der Laan etal, (1997) Tetrahedron Lett. 38:2249-52; U.S. Pat. No.6,063,569). PNA-DNA chimeras are designed to have desirable propertiesfound in PNA and DNA, e.g. superior hybridization properties of PNA andbiological functions like DNA (E. Uhlmann (1998) Biol. Chem.379:1045-52).

V.3 Structure of the Probes—a Second Portion for Binding to the OtherProbe(s)

The first probe also includes a second portion capable of hybridizing,directly or indirectly, with a second portion of a second probe. In thepreferred embodiment depicted in FIG. 1A, the association between thesecond portion 26 of the first probe 12 and the second portion 28 of thesecond probe 14 is a direct association by hybridization. In otherembodiments, the association between the second portions 26 and 28 isindirect. In these embodiments, one or more bridging molecules mayconnect the second portions of the probes. For example, as shown in FIG.1B, a bridging probe 29 hybridizes simultaneously with second portion 26and second portion 28 to form a four-way junction. Alternatively, abridging probe could associate simultaneously with second portion 26 andwith a second bridging probe capable of hybridizing with second portion28. In preferred embodiments, however, the association between secondportion 26 and second portion 28 is direct.

Both second portion 26 and second portion 28 preferably include anucleic acid and/or a nucleic acid analog. In more preferredembodiments, both second portion 26 and second portion 28 include ahigh-affinity nucleic acid analog such as PNA. Because PNA-PNA duplexesare more stable than PNA-DNA duplexes, as discussed above, use of PNAfavors the (appropriate) association of the probes with each otherrather than an (inappropriate) interaction between the second portionsof the probes and, for example, the target nucleic acid. Inappropriateinteractions between the second portions of the probes and the targetnucleic acid can be further disfavored by the use of sequences that arenot complementary to natural nucleic acids. For example, the use ofisocytosine-isoguanine base pairs between the second portions of theprobes would strongly disfavor inappropriate direct interaction betweena second portion and a natural target nucleic acid becausenaturally-occurring sequences do not comprise isocytosine or isoguanineand the isocytosine or isoguanine base pair is highly specific (U.S.Pat. Nos. 5,432,272 and 6,001,983; Tetrahedron Letters 36:3601-04).Other non-natural nucleobases could be used to the same effect (Fasman(1989) Practical Handbook of Biochemistry and Molecular Biology, pp.385-394, CRC Press, Boca Raton, Fla., as could other means to induce aspecific interaction, e.g. ligand-receptor, antigen-antibody, and thelike.

In most embodiments, the association occurs through noncovalent chemicalinteractions, such as hydrogen bonds (e.g. Watson-Crick orHoogsteen-type base pairing), ionic bonds, and/or hydrophobic forces.Although the probes are associated with each other when they areassociated with the target nucleic acid, in preferred embodiments theyare also associated with each other in solution in the absence of atarget nucleic acid. It is understood that the association of the probeswill be affected by environmental factors. These factors may include,for example, temperature, pH, ionic concentrations, and a variety ofother agents and factors well known in the art that influencehybridization. The practice of this invention requires only that theprobes comprise a chemical moiety that promotes the interaction of theprobes. By virtue of this tendency to associate, the binding of theprobes to a target nucleic acid is rendered cooperative.

V.4 Structure of the Probes—a Flexible Linker Joining the First andSecond Portions

The first and second portions of the first probe are typically joined bya flexible linker. In certain embodiments, a probe may not require aflexible linker. In the preferred embodiment depicted in FIG. 1A, thefirst portion 16 and the second portion 26 of first probe 12 are joinedby flexible linker 30. In this preferred embodiment, the first portion22 and second portion 28 of second probe 14 are also separated by aflexible linker 30. The flexible linker typically is a multi-atomlinker. The linker may optionally include one or more heteroatoms, anO-linker, and/or one or more ethyleneoxy units, —(CH₂CH₂O)—. Flexiblelinkers containing ethyleneoxy units are preferred where there are up tosix ethyleneoxy units. Ethyleneoxy linkage units can be linked to PNA orDNA moieties, for example, through amide or phosphate bonds. Ethyleneoxylinkage units can be installed by methods known in the art, for example,using coupling reagents such as protected forms of 2-[2-(2-aminoethoxy)ethoxy]acetic acid. The O-linker, 2-[2-(2-aminoethoxy)ethoxy]aceticacid, may be coupled as the FMOC-amino protected amide-formingcarboxylic acid, or phosphoramidite synthons. One or more O-linker unitsact as a flexible, non-base pairing linkage between the first and secondportions of the probe. The flexible linker may also be, for example, analkyldiyl consisting of 1 to 20 carbon atoms, such as hexyldiyl (U.S.Pat. No. 5,281,701). The linker could also be an aryldiyl consisting of6 to 20 carbon atoms, such as 1,4-phenyldiyl.

V.5 Structure of the Probes—Labeling Moieties

One or more of the probes may optionally include a detectable label. Inone embodiment, one or more of the probes is directly or indirectlyassociated with an enzyme having a detectable activity. In anotherembodiment, one or more of the probes comprises a moiety that canspecifically bind a detectable target. For example, a probe couldcomprise a biotin moiety, capable of specific binding to adetectably-labeled avidin. Alternatively, a probe could comprise anantigen, capable of specific binding to a detectable antibody. In apreferred embodiment, one probe comprises a fluorescent dye moiety, andone probe comprises a quenching moiety.

Labeling can be accomplished using any one of a large number of knowntechniques employing known labels, linkages, linking groups, reagents,reaction conditions, and analysis and purification methods. Nucleicacids and nucleic acid analogs can be labeled at sites including anucleobase, a sugar, the aminoethylglycine backbone, an amino residue, asulfide residue, a hydroxyl residue, or a carboxyl residue. Nucleobaselabel sites include the deaza C-7 or C-8 positions of the purine and theC-5 position of the pyrimidines. Preferably, the linkage between thelabel and the nucleic acid or nucleic acid analog is an acetylenic amidoor alkenic amido linkage (U.S. Pat. Nos. 5,770,716 and 5,821,356). Alinker can also comprise an alkyldiyl, aryldiyl, or ethyleneoxy unit.Typically, a carboxyl group on the label is activated by forming anactive ester, e.g., N-hydroxysuccinimide ester, and reacted with anamino group on the aminoethyleneoxy-, alkynylamino- oralkenylamino-derivatized nucleic acid or nucleic acid analog.Preferably, the nucleic acid or nucleic acid analog is anaminoethylenoxy derivative.

A nucleobase-labelled oligonucleotide may have the following formula:

where the oligonucleotide comprises 2 to 100 nucleotides. DYE is afluorescent dye, including energy transfer dye. B is a nucleobase, e.g.uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and8-deazaguanosine. L is a linker. R²¹ is H, OH, halide, azide, amine,C₁-C₆ aminoalkyl, C₁-C₆ alkyl, allyl, C₁ 14 C₆ alkoxy, OCH₃, orOCH₂CH═CH₂. R²² is H, phosphate, internucleotide phosphodiester, orinternucleotide analog. R²³ is H, phosphate, internucleotidephosphodiester, or internucleotide analog. In this embodiment, thenucleobase-labelled oligonucleotide may bear multiple fluorescentlabels, e.g. dyes, attached through the nucleobases. Nucleobase-labelledoligonucleotides may be formed by: (i) enzymatic incorporation ofenzymatically incorporatable nucleotide reagents where R¹⁹ istriphosphate, by a DNA polymerase or ligase, and (ii) coupling of anucleoside phosphoramidite reagent by automated synthesis. Whereas,nucleobase-labelled oligonucleotides may be multiply labelled byincorporation of more than one incorporatable nucleotide, labelling witha phosphoramidite dye label reagent leads to singly 5′-labelledoligonucleotides, according to the following formula:

where X is O, NH, or S; R²¹ is H, OH, halide, azide, amine, C₁-C₆aminoalkyl, C₁-C₆ alkyl, allyl, C₁-C₆ alkoxy, OCH₃, or OCH₂CH═CH₂; R²²is H, phosphate, internucleotide phosphodiester, or internucleotideanalog; and R²³ is H, phosphate, internucleotide phosphodiester, orinternucleotide analog. L is alkyldiyl, aryldiyl, or polyethyleneoxy.

Preferably, L is n-hexyldiyl.

A preferred class of labels provides a signal for detection of labeledextension products by fluorescence, chemiluminescence, andelectrochemical luminescence. Particularly preferred chemiluminescentlabels are 1,2-dioxetane compounds. Useful fluorescent dyes includefluoresceins, rhodamines, cyanines, and metal porphyrin complexes.

Examples of fluorescein dyes include those shown in FIGS. 2A and 2B(U.S. Pat. Nos. 5,366,860; 5,840,999; 6,008,379; 6,020,481; 5,936,087and 6,051,719). The 5-carboxyl, and other regio-isomers, may also haveuseful detection properties. Fluorescein and rhodamine dyes with1,4-dichloro substituents (bottom ring as shown) are especiallypreferred (U.S. Pat. Nos. 5,188,934; 5,654,442; 5,885,778; 5,847,162;6,025,505).

Another preferred class of labels includes fluorescence quenchers. Theemission spectrum of a quencher overlaps with a proximal intramolecularor intermolecular fluorescent dye such that the fluorescence of thefluorescent dye is substantially diminished or quenched by thephenomenon of fluorescence resonance energy transfer (FRET).

Particularly preferred quenchers include, but are not limited to,rhodamine fluorescent dyes including tetramethyl-6-carboxyrhodamine(TAMRA), tetrapropano-6-carboxyrhodamine (ROX), and DABSYL, DABCYL,asymmetric cyanines (U.S. Pat. No. 6,080,868), anthraquinone, malachitegreen, nitrothiazole, and nitroimidazole compounds and the like (FIG.3). Nitro-substituted forms of quenchers are especially preferred.

Energy-transfer dyes are another preferred class of oligonucleotidelabels. An energy-transfer dye label includes a donor dye linked to anacceptor dye or an intramolecular FRET pair (FIG. 2B). Light, e.g., froma laser, at a first wavelength is absorbed by a donor dye. The donor dyeemits excitation energy absorbed by the acceptor dye. The acceptor dyefluoresces at a second wavelength, with an emission maximum preferablyabout 100 nm greater than the absorbance maximum of the donor dye.

The donor dye and acceptor dye of an energy-transfer label may bedirectly attached by a linkage such as one formed from an aminomethylgroup at the 4′ or 5′ position of a donor dye and a 5- or 6- carboxylgroup of an acceptor dye (FIG. 3B). Other rigid and non-rigid linkersmay be useful.

Oligonucleotides that are intramolecularly labelled with bothfluorescent dye and quencher moieties are useful in nucleic acidhybridization assays, e.g. the “Taqman™” exonuclease-cleavage PCR assay(U.S. Pat. Nos. 5,538,848 and 5,723,591). In a Taqman™-type assay, theprobe is self-quenching, containing fluorescent dye and quenchermoieties. Spectral overlap allows for efficient energy transfer (FRET)when the probe is intact (Clegg, R. “Fluorescence resonance energytransfer and nucleic acids”, (1992) Meth. Enzymol, 211:353-388). Whenhybridized to a target, the probe is cleaved during PCR to release afluorescent signal that is proportional to the amount of target-probehybrid present.

In one preferred embodiment depicted in FIGS. 4A and 4B, a first probe40 comprises a fluorescent moiety 32 near one end and a quenching moiety34 near the other end. In this embodiment, a small region ofcomplementarity near the ends of the probe promotes the formation of ahairpin loop in the probe when the first probe is not associated with atarget nucleic acid, as depicted in FIG. 4A. The hairpin loop brings thequenching moiety 34 into proximity with the fluorescent moiety 32,quenching its fluorescence. When the probe participates in a complexwith a second probe 42 to form a three-way junction with a targetnucleic acid, the quenching moiety 34 is separated from the fluorescentmoiety 32, leading to fluorescence of the label.

A more preferred embodiment is depicted in FIGS. 4C and 4D. In thisembodiment, one probe 44 comprises a fluorescent moiety 32 near the endcomprising the portion that may be complementary to a target nucleicacid, and a second probe 46 comprises a quenching moiety 34 near the endcomprising the portion that may be complementary to a target nucleicacid. In the absence of the target nucleic acid, the fluorescent moiety32 and the quenching moiety 34 are held in proximity by a small regionof complementarity as shown in FIG. 4C. In the presence of the targetnucleic acid, the fluorescent moiety 32 and quenching moiety 34 areseparated, permitting fluorescence and detection of the complex.

In certain embodiments, different probes are labelled with different,independently detectable labels, such as fluorescent dyes havingspectrally-resolvable emission spectra to allow the simultaneousdetection of a plurality of target nucleic acid sequences. Suchmulti-label systems are advantageous in applications requiring analysisof multiple probe experiments. In such systems when the labels arefluorescent dyes, each dye can be identified by spectral resolution,enabling multiple target identification (U.S. Pat. Nos. 5,188,934;5,366,860; and 5,538,848).

V.6 Association With a Target Nucleic Acid

The target nucleic acid can be any nucleic acid or nucleic acid analogcapable of hybridizing to a primer or probe, or capable of mediatingtemplate-directed nucleic acid synthesis. As shown in the preferredembodiment in FIG. 1A, the binding moiety 10 contacts the target nucleicacid 20 through the first portions 16 and 22 of the first and secondprobes, 12 and 14, respectively. It should be noted that the firstportions 16 and 22 can associate with the same strand of the targetnucleic acid 20, as shown in FIG. 1A.

Where a first portion 16 or 22 includes PNA, the PNA should be orientedto permit an essentially antiparallel conformation between the nucleicacid of the first portion and the target nucleic acid 20. Theanti-parallel orientation (where the carboxyl terminus of PNA is alignedwith the 5′ terminus of the nucleic acid, and the amino terminus of PNAis aligned with the 3′ terminus of the nucleic acid) is preferredbecause the resulting complex is typically more stable (M. Egholm etal.,(1993) “PNA hybridizes to complementary oligonucleotides obeying theWatson-Crick hydrogen bonding rules,” Nature 365:566-68).

In one preferred embodiment, the binding of the probes to the targetnucleic acid leads to the formation of a three-way junction. As depictedin FIG. 1A, the flexible linker 30 of first probe 12 helps to alleviatethe strain associated with the three-way junction structure. Theflexible linker 30 of second probe 14, if present, also helps toalleviate the strain associated with the complex.

In a preferred embodiment, the first region 18 and the second region 24of the target nucleic acid 20 are substantially adjacent, i.e. separatedby zero or one nucleotides. This permits cooperativity from favorablebase-stacking interactions in addition to the cooperativity derived fromthe interaction between the second portions of the probes (Kandimallaetal (1995) Nucleic Acids Res. 23: 3578-84).

In a particularly preferred embodiment, the binding moiety includes twoPNA probes, each of which includes a flexible linker, and the bindingmoiety binds to a target nucleic acid to form a three-way junction. Ithas been discovered that, in this embodiment, efficient hybridization ispossible where one probe is complementary to a five nucleotide region ofa target nucleic acid and the other probe is complementary to a sevennucleotide region. Thus, in a preferred embodiment, one probe comprisesa first portion that may be complementary to a five nucleotide region ofa target nucleic acid, and a second probe comprises a first portion thatmay be complementary to a seven nucleotide region of a target nucleicacid. In another preferred embodiment, each of two probes comprises afirst portion that may be complementary to a six nucleotide region of atarget nucleic acid. However, it should be understood that smaller orlarger nucleotide regions may be encompassed by the invention.

The skilled artisan will appreciate that the invention is not limited tostructures containing three-way junctions. Complexes comprising variousbridging molecules could form a four-way junction as shown in FIG. 1B, afive-way junction, or another higher-order complex. One or more triplehelices (Kandimalla, etal (1995) Nucleic Acids Res. 23:4510-17;Kandimalla, etal (1995) Nucleic Acids Res. 23:1068-74) could be includedin the complex, as could a variety of double-helical conformations, suchas those resembling A-DNA, B-DNA, or Z-DNA. Additionally, the aboveelements may be combined in any operable combination. For example, oneembodiment of the invention is a structure with two three-way junctionsas shown in FIG. 1C. In this embodiment, a first probe 12 and a secondprobe 14 a form a three-way junction with the target nucleic acid 20,and the second probe 14 a and a third probe 35 also form a three-wayjunction with the target nucleic acid 20. In this embodiment, the secondprobe 14 a includes at least three portions: a first portion 28 acapable of hybridizing with the first probe, a second portion 22 acapable of hybridizing with the target nucleic acid, and a third portion28 b capable of hybridizing with the third probe. Many other geometriesare similarly envisioned. Only two elements of the geometry arerequired: first, that at least two probes interact directly orindirectly through their second portions 26 and 28; and second, that theprobes interact directly with the target nucleic acid (through firstportions 16 and 22).

V.7 Libraries (Arrays) of Probes

In another embodiment, the invention relates to libraries of probes usedto generate the above-described binding moieties. In this embodiment,the first portions of each of the first probes are complementary to adifferent sequence, and the first portions of each of the second probesare complementary to a different sequence. In a preferred embodiment,each of the second portions of each of the first probes is capable ofhybridizing with any of the second portions of each of the secondprobes. Thus, as shown in FIG. 5, the skilled artisan can select one ofthe first probes 12 from a first array 36 of a plurality of first probesand one of the second probes 14 from a second array 38 of a plurality ofsecond probes to generate a particular binding moiety 10.

Through judicious selection of a first probe and a second probe, theskilled artisan can generate a particular binding moiety complementaryto a particular nucleotide sequence of interest. The theory underlyingthe selection process is depicted in FIG. 6. In FIG. 6, the skilledartisan is provided with a first array 36 a of first probes. Forsimplicity in disclosing the general concept, and merely as exemplary,the first probes in FIG. 6 are each complementary to a dinucleotidesequence. It should be noted that in the actual practice of theinvention, however, each of the first probes would be complementary to asequence typically of from three to eight nucleotides in length. Thecomplete array of dinucleotide sequences includes sixteen probes, eachof which is complementary to a different nucleotide sequence. Theposition of each first probe in FIG. 6 is marked with the sequence ofits first portion, although it should be realized that each of the firstprobes also has an identical second portion complementary to a secondportion of the second probes in their respective array 38 a. Similarly,the skilled artisan is provided with a second array 38 a of secondprobes, each of which is complementary to a different dinucleotidesequence. The position of each second probe 14 is marked with thesequence of its first portion 22.

Continuing to refer to FIG. 6, to generate a binding moietycomplementary to the sequence “CGGA”, the skilled artisan selects probesfor a binding moiety with nucleobases “TCCG”. The skilled artisanselects from the first array 36 a a first probe containing “TC” andselects from the second array 38 a a second probe containing “CG” toform binding moiety 10 a which together with target may form a three-wayjunction. Alternatively, if the skilled artisan wishes to generate abinding moiety containing “AAGT”, the complement of the sequence “ACTT”,the artisan would select a first probe containing “AA” and a secondprobe containing “GT”. Thus, from two arrays of sixteen probes each, theartisan is provided with 256 possible binding moieties, one of which is10 a.

Accordingly, practice of the invention permits the skilled practitionerto use two or more low-complexity libraries to generate a probe, whereasa single high-complexity library would otherwise be necessary. Forexample, to generate a probe complementary to any twelve nucleotideregion of a target nucleic acid without practicing this invention, oneskilled in the art would require a library of 4¹² or 16,777,216 probes.In contrast, when practicing this invention, the skilled practitionerinstead could use a pair of libraries recognizing six nucleotides each,such that each library contained only 4⁶=4096 probes. Accordingly, inthis example, the practice of this invention requires the presence ofonly 2×4096=8192 probes, which is less than 0.05% (8192÷16,777,216) ofthe number of probes that would otherwise be required. Alternatively,when practicing the invention the skilled practitioner could use threelibraries recognizing four nucleotides each, such that each librarycontained only 4⁴=256 probes. The skilled artisan practicing theinvention would thus require only 3×256=768 probes, less than 0.005%(768÷16,777,216) of the number of probes required without practicing theinvention. Accordingly, the arrays of the present invention dramaticallyreduce the cost associated with producing, storing, and maintainingappropriate probe libraries.

An array of a plurality of probes preferably includes at least one probecapable of hybridizing to each possible first region of interest. Forexample, in FIG. 6, each first probe in the first array 36 a was capableof hybridizing to one particular dinucleotide sequence. The first arrayincluded 4² or 16 first probes, such that for any particular firstregion of interest, a probe was available to hybridize to it. Ingeneral, if each probe is complementary only to one particularnucleotide sequence of length x, the array should preferably include4^(x) probes. Alternatively, a probe may be complementary to more thanone particular nucleotide sequence, perhaps through the inclusion of oneor more “universal” nucleobases capable of hybridizing to any of aplurality of naturally-occurring nucleobases in a target nucleic acid.Thus, more generally, the array preferably includes 4^(x)÷N probes,where N is the number of nucleotide sequences to which each probe iscomplementary, i.e. the degeneracy of the probe.

Accordingly, in one embodiment, a first array includes at least0.5×4^(x)÷N probes, where x is the length, in nucleotides, of the firstregion and where N is the degeneracy of the first probes (the number ofnucleotide sequences to which each first probe is complementary).Preferably, in addition, a second array includes at least 0.5×4^(y)÷Mprobes, where y is the length, in nucleotides, of the second region andwhere M is the degeneracy of the second probes. More preferably, thefirst array includes at least 0.5×4^(x)÷N first probes and the secondarray includes at least 0.5×4^(Y)÷M second probes. More preferably, x+yis from 6 to 12.

The skilled artisan will appreciate that the arrays of the presentinvention lend themselves to embodiments in which the selection ofprobes is partially or fully automated. Thus, for example, either thetarget nucleic acid sequence or its complement could be provided to acomputer running a suitable software program. The software could thenanalyze the sequence and recommend one or more combinations of probessuitable for generating useful binding moieties. A software programcould also retrieve the desired probes robotically, and may mix theprobes in an appropriate combination. The software program for selectingthe probes could be the same as the software program for manipulatingthe probes, or the programs could be partially or completely independentfrom each other. The software could incorporate other optional featuresas well, such as the ability to accept and store shipping or billinginformation, the ability to label the probes for shipping to an enduser, or the ability to be accessed over the internet. Methods ofbuilding computerized systems capable of performing defined operationsare well known in the art and need not be elaborated herein. Thus, insome embodiments, the selection and preparation of probes is fullyautomated. In other embodiments, the selection and preparation are doneby humans with or without assistance from computerized systems.

V.8 Use of the Probes in Hybridizing to a Target Nucleic Acid

The binding moieties of the present invention are formed by combining atleast a first probe and a second probe in solution. It is understoodthat one of the probes may optionally be immobilized on a solid supportthrough an ionic interaction, affinity/receptor interaction, or covalentlinkage (U.S. Pat. No. 5,639,609). The solid substrate may be particles,beads, membranes, frits, slides, plates, micromachined chips,alkanethiol-gold layers, non-porous surfaces, or otherpolynucleotide-immobilizing media. The solid substrate material may bepolystyrene, controlled-pore-glass, silica gel, silica, polyacrylamide,magnetic beads, polyacrylate, hydroxyethylmethacrylate, polyamide,polyethylene, polyethyleneoxy, and copolymers and grafts of such. Inanother embodiment, the target nucleic acid may optionally beimmobilized on a solid substrate of the same configurations andmaterials (U.S. Pat. No. 5,821,060).

The binding moieties can be used as other nucleic acids or nucleic acidanalogs are used in the art. Thus, for example, the binding moieties canbe used to hybridize to a ribonucleic acid (RNA) to modulate theexpression and/or stability of the RNA. Modulation of RNA levels orexpression can be useful in medical science, agricultural science, andin a wide variety of biotechnological processes.

The binding moieties can also be used to detect a nucleotide sequence ofa nucleic acid or a nucleic acid analog. Detection could beaccomplished, for example, during gel electrophoresis of nucleic acidsor nucleic acid analogs by providing a detectable binding moiety withinthe gel. Detection could be accomplished following gel electrophoresisand transfer to a blot (e.g. nitrocellulose or nylon) by contacting theblot with a solution including a detectable binding moiety. Detectioncould be accomplished in situ in a cell or a tissue by contacting thecell or tissue with a solution including a detectable binding moiety.Detection could be accomplished in solution by observing changes in thephysico-chemical properties of the target nucleic acid or nucleic acidanalog after exposure to a binding moiety. In a preferred embodiment,detection of the binding event is facilitated by the presence of adetectable moiety, such as a fluorescent moiety, on at least one of theprobes.

Because the invention provides the skilled artisan a means to generate awide variety of binding moieties quickly and efficiently, many bindingmoieties may be used to analyze a sequence of interest. Thus, forexample, a variety of binding moieties complementary to a particular RNAcan be screened for their effects on the translation of the RNA. Nucleicacids could be screened for the presence of polymorphisms (e.g. singlenucleotide polymorphisms) or mutations. Once a particular sequence ofinterest has been identified, further analysis is possible using thebinding moieties of the present invention or using the oligonucleotideprobes of the prior art.

Exposure of the target nucleic acid to the binding moiety may optionallyoccur in the presence of a hybridization-stabilizing moiety such as aminor groove binder, an intercalator, a polycation such as poly-lysineor spermine, or a cross-linking functional group.Hybridization-stabilizing moieties may increase the stability ofbase-pairing (affinity), or the rate of hybridization, exemplified byhigh thermal melting temperatures (T_(m)) of the duplex. Hybridizationstabilizing moieties serve to increase the specificity of base-pairing,exemplified by large differences in T_(m) between perfectlycomplementary oligonucleotide and target sequences and where theresulting duplex contains one or more mismatches of Watson/Crick orHoogsteen-type base-pairing (“DNA and RNA structure” in Nucleic Acids inChemistry and Biology, (1996) G. Blackburn and M. Gait, eds., 2^(nd)edition, Oxford University Press, pp. 15-81). A preferred minor groovebinder is CDPI₃ (E. Lukhtanov etal., WO 96/32496; Lukhtanov etal (1995)Bioconjugate Chem. 6:418-26).

V.9 Use of Multipartite Binding Moieties in Priming Enzyme-CatalyzedReactions

The binding moieties can also be used as primers for enzyme-catalyzedreactions such as primer extension. When used as primers, the bindingmoieties should include at least a portion of a nucleic acid at theterminus of one of the probes. When the nucleic acid is present andhybridized to a complementary nucleic acid or nucleic acid analog, anappropriate enzyme (e.g. DNA-dependent DNA polymerase, RNA-dependent DNApolymerase, DNA-dependent RNA polymerase, and the like) can catalyze areaction for which the binding moiety serves as a primer.

Primer extension is initiated at the template site where a primeranneals. One or more different nucleotide 5′-triphosphates may bepresent in the reaction mixture such that the complementary nucleotideis incorporated by a polymerase enzyme according to the templatesequence. Extension of the primer continues until nucleotides aredepleted, the enzyme is no longer functional, or termination occurs byincorporation of a terminating nucleotide that will not supportcontinued DNA elongation. Chain-terminating nucleotides are typically2′,3′-dideoxynucleotide 5′-triphosphates (ddNTP) that lack the 3′-OHgroup necessary for 3′ to 5′ DNA chain elongation. Other terminatingnucleotides include 2′,3′-dideoxydehydro-; 2′-acetyl; 2′-deoxy, halo;and other 2′-substituted nucleotide 5′-triphosphates.

In general, the reaction conditions for primer extension involve anappropriate buffering system to maintain a constant pH. Also present area divalent cation, a binding moiety of the present invention, a targetnucleic acid, nucleotide 5′-triphosphates, and a polymerase. Additionalprimer extension reagents, such as reducing agents, monovalent cations,or detergents may be added to enhance the reaction rate, fidelity, orother parameters. Different polymerases may have different optimal pHvalues or ion concentrations. In some embodiments, a preferredpolymerase is thermostable. Kits useful in the practice of the inventionmay combine any or all of the above reagents.

Nucleotide 5′-triphosphates may be labelled for use in methods of theinvention. The sugar or nucleobase moieties of the nucleotides may belabelled. Preferred nucleobase labelling sites include the 8-C of apurine nucleobase, the 7-C or 8-C of a 7-deazapurine nucleobase, and the5-position of a pyrimidine nucleobase. The labelled nucleotide isenzymatically incorporatable and enzymatically extendable. Labellednucleotide 5′-triphosphates may have the following formula:

where DYE is a protected or unprotected dye, including energy transferdye. B is a nucleobase, e.g. uracil, thymine, cytosine, adenine,7-deazaadenine, guanine, and 8-deazaguanosine. R¹⁹ is triphosphate,thiophosphate, or phosphate ester analog. R²⁰ and R²¹, when taken alone,are each independently H, HO, and F. Linker L may be:

wherein n is 0, 1, or 2.

Labeled primer extension products, “fragments,” are generated throughtemplate-directed enzymatic synthesis using labeled binding moieties ornucleotides. The fragments can be separated by a size-dependent process,e.g., electrophoresis or chromatography, and the separated fragmentsdetected, e.g., by laser-induced fluorescence. In a preferred fragmentanalysis method, i.e., Sanger-type sequencing, a binding moiety isextended by a DNA polymerase in vitro using a single-stranded ordouble-stranded DNA template whose sequence is to be determined.Extension is initiated at a defined site based on where a binding moietyanneals to the template. The extension reaction is terminated byincorporation of a nucleotide that will not support continued DNAelongation, i.e., a terminating nucleotide. When optimizedconcentrations of dNTP and terminating nucleotides are used,enzyme-catalyzed polymerization (extension) will be terminated in afraction of the population of chains at each site where the terminatingnucleotide is incorporated such that a nested set of primer extensionfragments result. If fluorescent dye-labeled binding moieties or labeledterminating nucleotides are used for each reaction, the sequenceinformation can be detected by fluorescence after separation byhigh-resolution electrophoresis (U.S. Pat. No. 5,821,058). Each of thefour possible terminating nucleotides (A, G, C, T) may be present in theextension reaction and bear a different spectrally-resolvablefluorescent dye (U.S. Pat. No. 5,366,860).

The binding moieties of the present invention may also be used in“mini-sequencing,” another application involving incorporation ofterminating nucleotides to determine the identity, presence, or absenceof a nucleotide base at a specific position in a target nucleic acid(U.S. Pat. No. 5,888,819; A. Syvanen etal., (1990) “A primer-guidednucleotide incorporation assay in the genotyping of apolipoprotein E,”Genomics 8:684-92). Genotype determination based on identification ofdifferent alleles is based on single nucleotide polymorphisms (SNPs).SNPs can be detected by ddNTP incorporation from binding moietiesannealed immediately adjacent to the 3′ end of the SNP site of thetarget nucleic acid sequence to be determined, and detection of theextension products by MALDI-TOF mass spectroscopy. The mass differenceresulting from incorporation of different dideoxynucleotides can beaccurately determined by mass spectrometry. More than one bindingmoiety, with different sequences and masses, can be used in the samereaction to simultaneously detect multiple SNPs by analyzing the massspectra of the extension products (U.S. Pat. No. 5,885,775).

Primed in situ labeling (PRINS) is a molecular cytogenetic techniquethat combines the high sensitivity of PCR with the cellular orchromosomal localization of fluorescent signals provided by in situhybridization. PRINS can be conducted by annealing unlabeled bindingmoieties to complementary target nucleic acids, followed by a DNApolymerase extension in the presence of labeled dNTP. Preferably thelabels are fluorescent dyes, so that the extension products can bedetected and/or measured by fluorescence detection (J. Koch et a.,(1991) Genet. Anal. Tech. Appl. 8:171-78).

The invention is illustrated further by the following non-limitingexamples.

VI. EXAMPLES Example 1. Synthesis of Probes

PNA probes may be synthesized at any scale from commercially availablereagents and automated synthesizers, following the manufacturers'protocols. Most conveniently, PNA is synthesized at the 2 μmole scale,using Fmoc/Bhoc, tBoc/Z, or MMT protecting group monomers on an ExpediteSynthesizer (PE Biosystems) on XAL or PAL support, on the Model 433ASynthesizer (PE Biosystems) on MBHA support, or on other automatedsynthesizers. After synthesis is complete, the crude PNA is cleaved fromthe support, e.g. with trifluoroacetic acid, and then precipitated withdiethylether and washed twice in diethylether. PNA may be purified byreverse-phase HPLC, analyzed by mass spectroscopy, and quantitated bycorrelating absorbance at 260 nm with mass.

Oligonucleotide probes may be synthesized from commercially available(PE Biosystems) nucleoside phosphoramidites (U.S. Pat. No. 4,415,732)and solid supports, e.g. silica, controlled-pore-glass (U.S. Pat. No.4,458,066) and polystyrene (U.S. Pat. Nos. 5,047,524 and 5,262,530). Thephosphoramidite chemistry method of oligonucleotide synthesis isroutinely automated on commercially available synthesizers (Model 394DNA/RNA Synthesizer, PE Biosystems).

Labelling probes

Labelling typically results from mixing an appropriate reactive labeland a probe in a suitable solvent in which both are soluble, usingmethods well-known in the art (Hermanson, Bioconjugate Techniques,(1996) Academic Press, San Diego, Calif. pp. 40-55, 643-71), followed byseparation of the labelled probe from any starting materials or unwantedby-products. The labelled oligonucleotide can be stored dry or insolution for later use, preferably at low temperature.

The label may include a reactive linking group at one of the substituentpositions, e.g. 5- or 6-carboxy of fluorescein or rhodamine, forcovalent attachment to a probe. Reactive linking groups are moietiescapable of forming a covalent bond, typically electrophilic functionalgroups capable of reacting with nucleophilic groups on a probe, such asamines and thiols. Examples of reactive linking groups includesuccinimidyl ester, isothiocyanate, sulfonyl chloride, sulfonate ester,silyl halide, 2,6-dichlorotriazinyl, pentafluorophenyl ester,phosphoramidite, maleimide, haloacetyl, epoxide, alkylhalide, allylhalide, aldehyde, ketone, acylazide, anhydride, and iodoacetamide.

A preferred reactive linking group of a fluorescent dye is anN-hydroxysuccinimidyl ester (NHS) of a carboxyl group substituent of thefluorescent dye. The NHS ester of the dye may be preformed, isolated,purified, and/or characterized, or it may be formed in situ and reactedwith a nucleophilic group of an oligonucleotide. Typically, a carboxylform of the dye is activated by reacting with some combination of acarbodiimide reagent, e.g. dicyclohexylcarbodiimide,diisopropylcarbodiimide, or a uronium reagent, e.g. TSTU(O-(N-Succinimidyl) -N,N,N′,N′-tetramethyluronium tetrafluoroborate,HBTU (O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), or HATU(O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), an activator, such as 1-hydroxybenzotriazole(HOBt), and N-hydroxysuccinimide to give the NHS ester of the dye.

The linker between a probe and a label may be: (i) a covalent bond; (ii)an alkyldiyl —(CH₂)_(n)—, where n is 1 to 12; (iii) ethyleneoxy—(CH₂CH₂O)n—, where n is 1 to 12, (iv) aryldiyl (C₆ to C₂₀); or (v) oneor more amino acids. Lysine, aspartic acid, and glutamic acids arepreferred amino acid linkers in PNA probes. The sidechain, ε-amino groupof lysine may be the reactive linking group for attachment of a label,e.g. reporter dye or quencher. Linkers are typically attached to theamino and/or carboxyl terminus of the PNA by the corresponding monomerunits with compatible protecting groups and reactive functionality forcondensation with PNA monomer units and the solid support. For example,the O linker (2-[2-(2-aminoethoxy]acetic acid can be attached to theamino terminus of any PNA backbone amino group, or on aminofunctionality of a solid support. The 5′ hydroxyl terminus or anucleobase are preferred attachment sites on oligonucleotide probes.

PNA probe ME01 (CAGTCAGT-O-CCCAGCCTAT-Lys-Flu; wherein O denotes the Olinker, Lys denotes lysine and Flu refers to carboxyfluorescein and PNAprobe ME02 (Rho-O-ATAGCCCAGC-O-ACTGACTG; wherein Rho representstetramethylrhodamine) were synthesized on Expedite Nucleic AcidSynthesis System 8909 (PE Biosystems) employing commercially availableFmoc (Bhoc) monomers (PE Biosystems). The synthesis, labeling,purification and analysis were performed according to the manufacturer'sinstructions as described in “PNA synthesis for the Expedite NucleicAcid Synthesis System” (part number 601308), the teachings of which areherein incorporated by reference.

Example 2. Detection of a Target Nucleic Acid

PNA can hybridize to its target complement in either a parallel oranti-parallel orientation. However, the anti-parallel duplex (where thecarboxyl terminus of PNA is aligned with the 5′ terminus of DNA, and theamino terminus of PNA is aligned with the 3′ terminus of DNA) istypically more stable (Egholm, etal (1993) “PNA hybridizes tocomplementary oligonucleotides obeying the Watson-Crick hydrogen bondingrules”, Nature 365:566-68). The PNA FRET probes of the present inventionare designed such that the PNA anneals in the anti-parallel orientationwith the target sequences.

PNA molecules ME01 and ME02 were combined with DNA molecule 1057 as amodel target:

GGGCTGGGGCTGGGCAG (SEQ ID NO:1)

in 50 μL of 100 mM Tris-HCl, pH 8 to form the three-way junction shownin FIG. 7. The final concentration of each PNA and DNA molecule was 1μM. The mixtures were incubated at 95° C. for 10 minutes and graduallycooled to 37° C. over one hour in a PE GeneAmp PCR System 9700thermocycler (PE Biosystems). Five picomoles of the product were mixedwith a Hi-Density TBE (Tris(hydroxymethyl)aminomethane (Tris)-bufferedethylenediaminetetraacetic acid (EDTA)) sample buffer. The resultingsolution contained 45 mM Tris base, 45 mM boric acid, 0.4 mM EDTA, 3%(v/v) Ficoll, 0.02% bromophenol blue, and 0.02% xylene cyanol.

This solution, and others detailed below, was electrophoresed under bothnative (non-denaturing) and denaturing conditions. Native PAGE (FIG. 8)was conducted at room temperature and without urea in the gelformulation. Denaturing PAGE (FIG. 9) was conducted on a 10% denaturingPAGE gel (“15% TBE-urea gel”, Novex, San Diego, Calif.) at 35° C. andabout 130V for about 40 minutes. Both conditions included 10%polyacrylamide, run in 1×TBE (89 mM Tris base, 89 mM boric acid, 2 mMEDTA, pH 8.3. Samples were loaded onto the gels, electrophoresed, andvisualized by fluorescence detection under short wavelength UV light.

The presence of the annealed complex could be detected in lane 1 underboth native (FIG. 8) and denaturing (FIG. 9) conditions by fluorescenceof the carboxyfluorescein (Flu) and TAMRA (Rho) labels. The neutral PNAprobes, ME01 or ME02, do not migrate from the well duringelectrophoresis. The PNA-PNA-DNA complex (FIG. 7) was stable even underthe stringent denaturing conditions (FIG. 9). The complex required thepresence of both PNA molecules. If either was omitted, no complexformation was observed.

Example 3. Specificity of the Complex

PNA molecules ME01 and ME02 were combined either with the complementaryDNA molecule 1057, as above, or with DNA molecule 1058:

GGGCTGCCCTTTCTGGGCAG (SEQ ID NO. 2)

which bears a three nucleotide insertion (mut3bp) when compared to 1057,or with DNA molecule 1059:

GGGCTCGGGCTGGGCAG (SEQ ID NO. 3)

which bears a single nucleotide substitution (mut1bp). Annealing,electrophoresis and detection were carried out as described in Example2. Under native conditions, three-way junction complexes were formed foreach DNA (FIG. 8, lanes 1-3). Under denaturing conditions (FIG. 9),although the PNA-PNA-DNA complex was clearly evident when thecomplementary target sequence of 1057 was present (lane 1), no stablecomplex formation was observed in the presence of the mismatch of 1059(lane 2) or in the presence of the three nucleotide insertion of 1058(lane 3). The specificity of two PNA probes each with 7 base firstportions hybridizing to target and 8 base second portions hybridizing toeach other is thus demonstrated.

Example 4. Importance of the Second Portion of the Probe

PNA molecule ME01 was combined with DNA molecule 1057 in the presence ofeither PNA molecule ME02 or ME03 (Rho-O-ATAGCCCAGC-O), which lacks thesecond portion used by ME02 for interaction with ME01. Annealing wasperformed as described in Example 2. The annealing product was mixedwith sample buffer to a final concentration of 45 mM Tris base, 45 mMboric acid, 0.4 mM EDTA, 0.15g/mL Ficoll, and 0.07% xylene cyanol. Thesample was electrophoresed under the same native (FIG. 8, lane 4) anddenaturing (FIG. 9, lane 4) conditions as described in Example 2. Thesingle PNA probe ME02 with a 7 base first portion hybridizing to target1057 gave no observable duplex formation even under native conditions.

Although some ME01/ME03/1057 complex formation was observed (result notshown), complex formation was significantly reduced when compared to theME01/ME02/1057 complexes, despite the relatively permissive conditionsof the gel.

A single PNA probe 1133 (Rho-O-GCCCAGC-O-CCCAGCC) with 14 baseshybridizing to target was hybridized with each of the three targets;1057 (lane 5), 1059 (lane 6), and 1058 (lane 7) under native anddenaturing conditions. The PNA nucleobases in 1133 bind to target 1057with an O linker bulge in 1133, and additionally the C/G mismatch with1059. The duplex between 1133 and 1058 has the O linker bulge in 1133and a three base TTT bulge in 1058. Under native conditions, stableduplexes were observed with each of the three targets, but under themore stringent denaturing conditions, only the O linker bulge duplex inlane 5 formed a stable duplex.

A single PNA probe 1134 (Rho-O-GCCCAGCCCCAGCC) differs from 1133 only bythe absence of the O linker in the middle of the sequence. PNA probe1134 was also hybridized with each of the three targets; 1057 (lane 8),1059 (lane 9), and 1058 (lane 10) under native and denaturingconditions. The PNA nucleobases in 1134 bind to target 1057 as a perfectmatch, and to 1059 with a single mismatch. The duplex between 1134 and1058 has a three base TTT bulge in 1058. Under native conditions (FIG.8), approximately equally stable duplexes were observed with each of thethree targets. Under the more stringent denaturing conditions (FIG. 9),the perfect match (lane 8) showed the most intense band, althoughmismatch duplexes are observed in lanes 9 and 10. Therefore littlespecificity is observed in duplex formation with a single PNA probe with14 bases hybridizing to target. Thus, the second portions of two probes,which mediate the direct interaction between the PNA molecules, isimportant both to the stability of the tripartite complex and specificdetection of target nucleic acid.

PNA probes with shorter first portions binding to target wereinvestigated (lanes 11, 12, 13). Unlabelled PNA probe 1136(O-CAGTCAGT-O-CCCAG), rhodamine labelled PNA probe 1137(Rho-O-CCAGC-O-ACTGACTG) were annealed under the conditions ofExperiment 2 with perfect match target 1057 (lane 11), one base mismatchtarget 1059 (lane 12), and three base bulge 1058 (lane 13). Each samplewas electrophoresed under native (FIG. 8) and denaturing conditions(FIG. 9). Probe 1136 has a five base first portion hybridizing to targetand probe 1137 has a six base first portion hybridizing to target. Probe1137 has the rhodamine dye attached directly to the 5′ terminus of Cbase which binds to target and is without the non-binding sequence (ATA)present in probe ME02. Probes 1136 and 1137 hybridize to each other byan eight base second portion. No stable complexes are observed with thethree samples under even the native electrophoresis conditions.

PNA probes ME01 and 1137 were investigated for complex formation byannealing with targets 1057 (lane 14), 1058 (lane 15), and 1059 (lane16) under the conditions of Experiment 2. Samples were electrophoresedunder native (FIG. 8) and denaturing conditions (FIG. 9). Specificitywas observed under native conditions where only the perfect matchcomplex was observed (FIG. 8, lane 14). No duplex was observed with PNAprobe 1137 alone binding with perfect match target 1057 (lane 17).

PNA probes 1136 and ME02 were investigated for complex formation byannealing with targets 1057 (lane 18), 1058 (lane 19), and 1059 (lane20) under the conditions of Experiment 2. Specificity was observed undernative conditions where only the perfect match complex was observed(FIG. 8, lane 18).

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

Each of the patent documents and scientific publications disclosedhereinabove is incorporated by reference herein.

3 1 17 DNA Unknown Synthetic DNA 1 gggctggggc tgggcag 17 2 20 DNAUnknown Synthetic DNA 2 gggctgccct ttctgggcag 20 3 17 DNA UnknownSynthetic DNA 3 gggctcgggc tgggcag 17

What is claimed is:
 1. A first array of a plurality of single-strandedfirst probes and a second array of a plurality of single-stranded secondprobes, wherein the first probes comprise a first portion complementaryto a first region of a target nucleic acid and capable of hybridizingthereto, and a second portion capable of hybridizing with the secondprobe, wherein the first and second portions of the first probe arejoined by a flexible linker, wherein the flexible linker comprises 1 to6 ethyleneoxy units, alkyldiyl of 1 to 20 carbon atoms, or aryldiyl of 6to 20 carbons atoms; and the second probes comprise a first portioncomplementary to a second region of the target nucleic acid and capableof hybridizing thereto, and a second portion capable of hybridizing withthe first probe; the first region of the target nucleic acid and thesecond region of the target nucleic acid are substantially adjacent;each of the first and the second region of the target nucleic acid isfrom three to eight nucleotides in length; at least one of the firstprobe or the second probe comprises a high-affinity nucleic acid analog;at least one of the first probe or the second probe comprises afluorescent detection moiety; the portion of each of the first probescomplementary to the first region of the target nucleic acid has adifferent sequence, wherein the first region of the target nucleic acidis x nucleotides in length and x is greater than or equal to 6; thearray of first probes comprises at least (0.5×4^(x))÷N first probes,wherein N is equal to the degeneracy of each of the first portions ofeach of the first probes; and the portion of each of the second probescomplementary to the second region of the target nucleic acid has adifferent sequence.
 2. The first array and second array of claim 1wherein the second region of the target nucleic acid is y nucleotides inlength and y is greater than or equal to 6, and wherein the array ofsecond probes comprises at least (0.5×4^(y))÷M second probes, wherein Mis equal to the degeneracy of each of the first portions of each of thesecond probes.
 3. The first array and second array of claim 1 whereinthe second portion of a said first probe and the second portion of asaid second probe comprise regions which are complementary to eachother; and wherein the first and second portions of the second probe arejoined by a flexible linker, wherein the flexible linker comprises 1 to6 ethyleneoxy units, alkyldiyl of 1 to 20 carbon atoms, or aryldiyl of 6to 20 carbon atoms.
 4. The first array and second array of claim 1wherein the second portion of a said first probe hybridizes with agreater binding affinity with the second portion of a said second probethan with any portion of the target nucleic acid.
 5. The first array andsecond array of claims 1 wherein the portion of a said first probecomplementary to the first region of a target nucleic acid comprises ahigh-affinity nucleic acid analog.
 6. The first array and second arrayof claim 1 wherein the high-affinity nucleic acid analog comprises oneor more PNA monomer units.
 7. The first array and second array of claim6 wherein the peptide nucleic acid has a 2-aminoethylglycine backbone.8. The first array and second array of claim 1 wherein a probe comprisesa quenching moiety.
 9. The first array and second array of claim 1wherein a probe comprises an oligonucleotide sequence.
 10. The firstarray and second array of claim 1 wherein a said second probe furthercomprises a flexible linker selected from 1 to 6 ethyleneoxy units,alkyldiyl of 1 to 20 carbon atoms, and aryldiyl of 6 to 20 carbon atoms.11. The first array and second array of claim 1 wherein a probe isimmobilized on a solid support.
 12. The first array and second array ofclaim 1 wherein the target nucleic acid is immobilized on a solidsupport.
 13. The first array and second array of claim 1 wherein thehigh-affinity nucleic acid analog are selected from locked nucleicacids, 2′-O-methyl nucleic acids, and 2′-fluoro nucleic acids.
 14. Thefirst array and second array of claim 1 wherein the second portion of asaid first probe and the second portion of a said second probe comprisean isocytosine-isoguanine base pair.
 15. A kit comprising the firstarray of a plurality of first probes of claim 1 and a buffer.
 16. Thekit of claim 15 further comprising the second array of a plurality ofsecond probes.
 17. The kit of claim 15 further comprising an enzyme.